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. 2012 Aug;10(8):1120-32.
doi: 10.1158/1541-7786.MCR-12-0099. Epub 2012 Jun 5.

Ligand Binding Promotes CDK-dependent Phosphorylation of ER-alpha on Hinge Serine 294 but Inhibits Ligand-Independent Phosphorylation of Serine 305

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Ligand Binding Promotes CDK-dependent Phosphorylation of ER-alpha on Hinge Serine 294 but Inhibits Ligand-Independent Phosphorylation of Serine 305

Jason M Held et al. Mol Cancer Res. .
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Abstract

Phosphorylation of estrogen receptor-α (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic value as a biomarker for ERα-positive breast cancers. Recent attention has turned to the poorly understood ERα hinge domain, as phosphorylation at serine 305 (Ser305) associates with poor clinical outcome and endocrine resistance. We show that phosphorylation of a neighboring hinge domain site, Ser294, analyzed by multiple reaction monitoring mass spectrometry of ERα immunoprecipitates from human breast cancer cells is robustly phosphorylated exclusively by ligand (estradiol and tamoxifen) activation of ERα and not by growth factor stimulation (EGF, insulin, heregulin-β). In a reciprocal fashion, Ser305 phosphorylation is induced by growth factors but not ligand activation of ERα. Phosphorylation at Ser294 and Ser305 is suppressed upon co-stimulation by EGF and ligand, respectively, unlike the N-terminal (AF-1) domain Ser118 and Ser167 sites of ERα where phosphorylation is enhanced by ligand and growth factor co-stimulation. Inhibition of cyclin-dependent kinases (CDK) by roscovitine or SNS-032 suppresses ligand-activated Ser294 phosphorylation without affecting Ser118 or Ser104/Ser106 phosphorylation. Likewise, cell-free studies using recombinant ERα and specific cyclin-CDK complexes suggest that Ser294 phosphorylation is primarily induced by the transcription-regulating and cell-cycle-independent kinase CDK7. Thus, CDK-dependent phosphorylation at Ser294 differentiates ligand-dependent from ligand-independent activation of Ser305 phosphorylation, showing that hinge domain phosphorylation patterns uniquely inform on the various ERα activation mechanisms thought to underlie the biologic and clinical diversity of hormone-dependent breast cancers.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

D.J. Britton has employment (other than primary affiliation; e.g., consulting) in Proteome Sciences plc as Senior Research Scientist, has a commercial research grant from Proteome Sciences plc, and also has other commercial research support from Proteome Sciences plc. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1
Figure 1
MCF-7 cells transfected with an Ser294Ala-mutated ERα expression construct produce transcriptional suppression of E2-inducible genes. A, immunoblot analysis of ERα protein levels in MCF levels following transfection with empty vector, exogenous wild-type (WT) ERα, or Ser294Ala ERα in the absence (C) or presence (E2) of 10 nmol/L E2 for 6 hours. Note the similar degree of ERαdownregulation following E2 stimulation relative to basal levels in all 3 transfection sets. Equal protein loading was confirmed by probing for β-actin. B, quantitation of gene transcript levels by densitometry of PCR products visualized with ethidium. Levels of 3 E2-inducible genes (AREG, EGR3, CXCL12) are normalized relative to empty vector control levels (set equal to 1.0) with all gene transcripts levels originally normalized by their respective GAPDH. Error bars represent SD of densitometric quantifications obtained from 3 biologic replicates. *, significant reductions (P < 0.05) in the Ser294Ala inductions relative to E2-stimulated empty vector and WT ERα transfectants as assessed by the Student t test (2-way, unpaired). See Materials and Methods for specific gene primers.
Figure 2
Figure 2
Phosphorylation of ERα Ser294 is induced by E2, but not EGF, in MCF-7 cells. A, MRM/MS assay chromatograms for the phosphorylated and unmodified Ser294 and Ser167 peptides from trypsin-digested ERα immunoprecipitates from MCF-7 cells. The MRM/MS transitions used to quantify the Ser294 peptide, 288-AANLWPSPLMIK-299, were Q1/Q3 670.9/785.5 corresponding to the [M+2H]2+ precursor and y7 fragment ion for the unmodified peptide and for the phosphorylated peptide Q1/Q3 710.9/767.4 was used corresponding to the [M+2H]2+ precursor and y7-98 fragment ion. The MRM transitions used to quantify the Ser167 peptide, 165-LASTNDKGSMAMESAK-180, were Q1/Q3 547.6/764.3 corresponding to the [M+3H]3+ precursor and y152+ for the unmodified peptide and for the phosphorylated peptide Q1/Q3 574.2/755.3 was used corresponding to the [M+3H]3+ precursor and y152+-49 fragment ion. MCF-7 cells were treated with 10 nmol/L E2 for 30 minutes or 8 nmol/L EGF for 10 minutes. For EGF treatment, cells were serum-starved (NS) for 24 hours before treatment, and for E2, cells were grown in charcoal-stripped (CS) media for 24 hours before treatment. B, MRM peak areas were used to calculate the phosphorylated:unmodified peptide area ratio, the relative MRM/MS peak areas of the phosphorylated 167 and 294 peptides normalized to the MRM/MS peak area of the unmodified peptide for each condition in A. Error bars represent the SD of at least 4 biologic replicates. *, a significant difference (P < 0.05) between the treatments and respective controls as assessed by the Student t test (2-way, unpaired). C, Western blot analysis of pSer167 levels induced following E2 and EGF treatments similar to those described in A confirm the concordance of Western blotting results with the MRM pSer167 induction results shown in B. Ser167 phosphorylation is induced by E2 in CS, but not NS, growth conditions. D, determination of the maximal stoichiometry of Ser294 and Ser167 phosphorylation using SID-MRM. The percentages of phosphorylation of Ser294 and Ser167 are maximal under E2 and EGF treatment, respectively, which are shown. Cells were grown in serum-free conditions for 24 hours. Error bars represent the SD of 2 independent biologic replicates.
Figure 3
Figure 3
E2, but not EGF, induces phosphorylation of Ser294 in multiple cells lines. MRM/MS analysis of the induction of Ser294 phosphorylation by E2 and EGF in BT474, MCF-7, and T47D cell lines. A, in all cell lines, E2 induces Ser294 phosphorylation. B, EGF stimulation does not induce Ser294 phosphorylation in any cells line. Cells were treated with 8 nmol/L EGF for 10 minutes or 10 nmol/L E2 for 30 minutes. For EGF treatment, cells were serum-starved (NS) for 24 hours before treatment, and for E2, cells were grown in charcoal-stripped (CS) media for 24 hours before treatment. Error bars represent the SD of at least 3 biologic replicates. *, a significant change (P < 0.05) as assessed by the Student t test (2-way, unpaired). CTL, control; TAM, tamoxifen.
Figure 4
Figure 4
ERα hinge domain phosphorylation at Ser294 and Ser305 is suppressed by cross-talk between growth factor and ligand stimulation whereas N-terminal phosphorylation at pSer118 and pSer167 is enhanced. A, MCF-7 cells in serum-free conditions were either untreated (CTL), treated with E2 (10 nmol/L) for 30 minutes, EGF (8 nmol/L) for 14 minutes, or 10 μmol/L forskolin (Fors) for 14 minutes. B, Western blot analysis of ER immunoprecipitated from MCF-7 cells either untreated (—), treated with EGF (8 nmol/L) for 20 minutes, treatedwithE2(10nmol/L) for 20 minutes, or cotreated with EGF (8 nmol/L) and E2 (10, 2, and 0.2 nmol/L) for 20 minutes. Total ERα served to normalize lane loading. C, MRM/MS analysis of immunoprecipitated/trypsin-digested ER from MCF-7 cells; untreated (CTL), E2 (10 nmol/L)-, EGF (8 nmol/L)-, and E2 + EGF (10 nmol/L E2, 8 nmol/L EGF)-treatedsamplesshowninBwiththe MRM/MS peak area ratio used to determine the induction of Ser294 and Ser167 phosphorylation. Percentage of E2 induction of Ser294 and percentage of EGF induction of pSer167 are shown. *, P value below 0.05 as assessed by the Student t test (2-way, unpaired).
Figure 5
Figure 5
CDKs inhibit Ser294 phosphorylation in vivo and can phosphorylate Ser294 in vitro. A, in vitro phosphorylation of Ser294 CDKs, including CDKs 2, 4, 7, and 9 examined by Western blot analysis using pSer294 antibody. B, MRM/MS analysis of Ser294 phosphorylation of ER immunoprecipitated from MCF-7 cells treated with 10 nmol/L estradiol (E2) for 30 minutes or first pretreated for 1 hour with the broad-spectrum CDK inhibitors roscovitine (5 and 50 μmol/L) or SNS-032 (1 μmol/L) before E2 treatment. Both inhibitors decrease Ser294 phosphorylation relative to E2 alone (set equal to 100%). Error bars represent the SD for 3 biologic replicates. C, Western blot analysis of immunoprecipitated ER also showed a decrease in Ser294 phosphorylation following CDK inhibitor treatment using a pSer294 antibody. Western blot analysis of immunoprecipitated ER using antibodies to pSer118 and pSer104/pSer106 showed that inhibition of CDKs does not reduce their induction by E2. Equal ER loading is confirmed by probing for ERα.
Figure 6
Figure 6
A model of ERα phosphorylation under ligand and growth factor stimuli. ERα (purple) consists of an AF-1, DNA-binding domain (DBD), hinge, and LBD/AF-2 domains. Ser118 phosphorylation is induced by both ligand (blue arrow) and growth factor stimulation (green arrow), whereas Ser167 phosphorylation is primarily induced by growth factors (green arrow) although weakly induced by ligand (dashed blue arrow). Kinases shown promoting pSer118 and pSer167 are not meant to exclude other AF-1 phosphorylating candidates; all candidate kinases as well as the indicated kinases promoting pSer305 are described in the work of Le Romancer and colleagues (2) except the ligand stimulation of pSer167 found in Britton and colleagues (27) and results shown in Fig. 2C. In the hinge domain, our study determined that Ser294 is phosphorylated by a CDK that is exclusively ligand-induced. Ser305 is induced only by growth factors. In contrast to the phosphorylation sites in the AF-1 domain which are co-stimulated by ligand and growth factors, hinge domain phosphorylation at Ser294 and Ser305 is suppressed by co-stimulation with ligand and growth factors (red lines) with the heavier red line to pSer305 relative to the thinner red line to pSer294 indicating the approximately 5-fold greater suppression of pSer305 relative to pSer294 at the co-stimulatory concentration of 10 nmol/L estrogen with 8 nmol/L EGF (see Fig. 4A). MAPK, mitogen-activated protein kinase.

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